Capillary malformation-arteriovenous malformation (CM-AVM) is an autosomal dominant vascular disorder characterized by CM and life-threatening AVM and arteriovenous fistulas. Germline inactivating mutations of the RASA1 gene, which encodes RASA1, a negative-regulator of the Ras signaling pathway, and of EPHB4, which encodes the EPHB4 growth factor receptor, have been identified as the cause of most cases of CM- AVM. This suggests that RASA1 and EPHB4 function in the same molecular pathway to protect against the development of blood vascular (BV) lesions in this disease. Studies of RASA1- and EPHB4-deficient mice indi- cate that both molecules are required for developmental angiogenesis and that defects in this process underlie the occurrence of BV abnormalities in CM-AVM. However, how RASA1 and EPHB4 promote developmental angiogenesis is unknown. A long-term goal of the King laboratory is to understand the role of the Ras signaling pathway in different physiological systems in health and disease. The overall objective of this application, which is consistent with this long-term goal, is to understand the molecular mechanisms involved in the devel- opment of BV lesions in CM-AVM. Our central hypothesis is that dysregulated Ras signaling in RASA1- and EPHB4-deficient BV endothelial cells (BEC) results in increased expression of the PLOD2 collagen IV- modifying enzyme that is primarily responsible for impaired folding of collagen IV, thus leading to its retention in BEC, which blocks developmental angiogenesis. Accordingly, we hypothesize that chemicals that assist col- lagen IV folding will prevent the development of CM-AVM lesions. The rationale of the proposed studies is that they will inform upon the molecular mechanisms involved in the etiology of BV lesions in CM-AVM, as well as possible means of prevention. We plan to test our central hypothesis and, thereby, attain the objective of this application by pursuing the following three specific aims: In the first aim, we will use different molecular biolog- ic, proteomic, and mouse genetic approaches to establish that in RASA1-deficient BEC dysregulated Ras sig- naling impairs collagen IV folding and export as a result of increased expression of PLOD2. In the second aim, we will employ similar approaches to determine if loss of EPHB4 impairs developmental angiogenesis through the same mechanism and if this is explained by an absence of physical interaction with RASA1. In the third aim, we will characterize a novel physiological mosaic RASA1-deficient mouse model of CM-AVM that will be used to test the ability of drugs that promote collagen IV folding to prevent the development of BV lesions in this disease. The proposed studies are innovative because of the novel mouse models of CM-AVM used and the unexpected discovery of the link between dysregulated Ras signaling and collagen IV folding and export. The studies are significant because of their potential to illuminate upon the molecular pathogenesis of CM- AVM and ways in which lesion development might be prevented in genetically susceptible individuals. !